Implantable compositions and methods for preparing the same

ABSTRACT

Methods for forming implantable compositions are provided. In some embodiments, the methods include (i) providing a gel base, (ii) adding water and a hydrating agent to the gel base to form a mixture, (iii) reducing the water content of the mixture; and (iv) adding a delivered material before, during, and/or after step (ii) or (iii). The water content is reduced to about 5% or less by weight of the implantable composition.

This application is a divisional application of U.S. patent applicationSer. No. 13/090,745 filed Apr. 20, 2011, entitled “IMPLANTABLECOMPOSITIONS AND METHODS FOR PREPARING THE SAME”. This entire disclosureis incorporated herein by reference into the present disclosure.

BACKGROUND

Overview of Grafts for Bone Repair

Implantable materials are used in a variety of applications, often torepair or replace tissue. Implantable materials are of particularinterest in the repair of bone defects. The rapid and effective repairof bone defects caused by injury, disease, wounds, or surgery has longbeen a goal of orthopaedic surgery. Toward this end, a number ofcompositions and implantable materials have been used or proposed foruse in the repair of bone defects. The biological, physical, andmechanical properties of the compositions and materials are among themajor factors influencing their suitability and performance in variousorthopaedic applications.

Autologous cancellous bone (“ACB”) long has been considered the goldstandard for bone grafts. ACB includes osteogenic cells, which have thepotential to assist in bone healing, is nonimmunogenic, and hasstructural and functional characteristics that should be appropriate fora healthy recipient. Some people do not have adequate amounts of ACB forharvesting. These people include, for example, older people and peoplewho have had previous surgeries. Further, there is reluctance to harvestACB because of pain at the harvest site and donor morbidity.

Much effort has been invested in the identification and development ofalternative bone graft materials. Urist has published seminal articleson the theory of bone induction and a method for decalcifying bone,i.e., making demineralized bone matrix (DBM). Urist M. R., BoneFormation by Autoinduction, Science 1965; 150(698):893-9; Urist M. R. etal., The Bone Induction Principle, Clin. Orthop. Rel. Res. 53:243-283,1967. DBM is an osteoinductive material, in that it induces bone growthwhen implanted in an ectopic site of a rodent, owing to theosteoinductive factors contained within the DBM. Honsawek et al. (2000).There are numerous osteoinductive factors, e.g., BMP 1-18, which arepart of the transforming growth factor-beta (TGF-beta) superfamily.BMP-2 has been widely studied. There are also other proteins present inDBM that are not osteoinductive alone but still contribute to bonegrowth, including fibroblast growth factor-2 (FGF-2), insulin-likegrowth factor-I and -II (IGF-I and IGF-II), platelet derived growthfactor (PDGF), and transforming growth factor-beta 1 (TGF-beta.1).(Hauschka, et al. 1986; Canalis, et al, 1988; Mohan et al. 1996.)

Various cocktails of growth factors have been measured in DBM, includingBMP2, TGFβ1, FGFa, IGF-I, PDGF, VEGF (Wildemann et al, 2007), BMP4 (Blumet al, 2004 and Honsaweket et al, 2005), and BMP7 (Pietrzak et al,2006). Other extracellular matrix proteins have also been measured,including type I collagen, fibronection, Bone Sialoprotein (BSP), andosteopontin (Shigeyama et al, 1995). Combinations of growth factors maybe more osteoinductive than a single growth factor (Kawai et al, 2006,Mehlhorn et al, 2007, Shintani et al, 2007, Raiche et al, 2004 andRipamonti et al, 1997). DBM extracellular matrix proteins such ascollagen may be used as carriers for the growth factors (Reddi et al,2000).

DBM implants have been reported to be particularly useful (see, forexample, U.S. Pat. Nos. 4,394,370, 4,440,750, 4,485,097, 4,678,470, and4,743,259; Mulliken et al., Calcif Tissue Int 33:71, 1981; Neigel etal., Opthal. Plast. Reconstr. Surg. 12:108, 1996; Whiteman et al., J.Hand. Surg. 18B:487, 1993; Xiaobo et al., Clin. Orthop. 293:360, 1993,each of which is incorporated herein by reference). Useful DBM implantsare disclosed in U.S. Pat. Nos. 5,073,373; 5,284,655; 5,290,558;5,314,476; 5,507,813; 5,510,396; and 5,676,146, each of which isincorporated by reference herein.

DBM may be derived from donated human tissue. The bone is removedaseptically and treated to kill any infectious agents. The bone istypically particulated by milling or grinding, and then the mineralcomponent is extracted by various methods, such as by soaking the bonein an acidic solution. The remaining matrix is malleable and can befurther processed and/or formed and shaped for implantation into aparticular site in the recipient.

In some grafts, non-bone materials may be used, includinghydroxyapatite, ceramics, calcium sulfate, calcium phosphates,tricalcium phosphate, bioactive glasses, other materials, andcombinations of these.

In some applications, the graft material (which also may be referred toas a delivered material), which may include biologics and/or non-bonematerials, are combined with a carrier. The delivered material-carriercombination then may be shaped into a suitable bone graft.

Carriers

Generally, a carrier may be employed to facilitate in vivo use and/orformation of a particular configuration of the implantable material. Inconsidering carriers for combination with a delivered material, it isdesirable to have a carrier that has minimal effect on bioactivecompounds and/or biocompatible compounds of the delivered material.Suitable carriers may include, for example, glycerol or hydrogel. Thespecific carrier used may be selected based on desired handlingcharacteristics and surgeon preference.

Hydrogels exhibit many properties that make them suitable carriers forimplantable materials used in the repair of bone and other tissuedefects. For example, hydrogels exhibit controlled viscosity over a widerange of values, strong resistance to surgical irrigation, and excellentbiocompatibility. Hydrogels are formed of a highly absorbent natural orsynthetic polymer base material dispersed in water. Water has been theuniversal solvent for hydrogels because those skilled in the art havenot perceived disadvantages to the use of water, and because nonaqueoussolvents for hydrogels have been thought impractical or impossible.

The inventors have determined that, while hydrogels may provide suitableor desirable handling characteristics to an implantable material, thepresence of water may have deleterious effects on the bioactivity of thematerial, and/or on the material's shelf life. Accordingly, a processthat adequately solubilizes or otherwise disperses the polymeric basematerial of a hydrogel in a nonaqueous solvent, or a water substitute,but which produces a material having similar or superior physical andbiologic properties to hydrogels as a carrier, is desirable. The havedeveloped, and disclose herein, a method for preparing a hydrogelcarrier that has a substantial absence of water.

SUMMARY

Methods for forming implantable compositions are provided herein. Insome embodiments, the methods include (i) providing a gel base, (ii)adding water and a hydrating agent to the gel base to form a mixture,(iii) reducing the water content of the mixture; and (iv) adding adelivered material before, during, and/or after step (ii) or (iii). Thewater content is reduced to about 5% or less by weight of theimplantable composition.

Implantable compositions are also provided. The implantable compositionsinclude a delivered material and a carrier. The carrier includes a gelbase and a hydrating agent. A water content of the implantablecomposition is less than about 5% by weight of the composition.

The implantable compositions may be formed and used in any of a varietyof desirable manners. For example, the implantable composition may beformed into an osteoimplant and applied at a bone repair site, or at asite for bone augmentation or ectopic bone formation (e.g., lateralspine fusion). Further examples of the use of the implantablecompositions disclosed herein include at bone or other tissue sites thathave been injured, or that have defects brought about during the courseof surgery, disease, trauma, malignancy, or developmental malformation.

This application refers to various patents, patent applications, journalarticles, and other publications, all of which are incorporated hereinby reference. The following documents are incorporated herein byreference: PCT/US04/43999; PCT/US05/003092; US 2003/0143258 A1;PCT/US02/32941; Current Protocols in Molecular Biology, CurrentProtocols in Immunology, Current Protocols in Protein Science, andCurrent Protocols in Cell Biology, John Wiley & Sons, N.Y., edition asof July 2002; Sambrook, Russell, and Sambrook, Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 2001; Rodd 1989 “Chemistry of Carbon Compounds,” vols.1-5 and supps., Elsevier Science Publishers, 1989; “Organic Reactions,”vols. 1-40, John Wiley and Sons, New York, N.Y., 1991; March 2001,“Advanced Organic Chemistry,” 5th ed. John Wiley and Sons, New York,N.Y. In the event of a conflict between the specification and any of theincorporated references, the specification shall control. Wherenumerical values herein are expressed as a range, endpoints areincluded.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed description is to be regarded as illustrativein nature and not restrictive.

BRIEF DESCRIPTION

FIG. 1 illustrates a method of making an implantable composition inaccordance with one embodiment of the present disclosure.

DEFINITIONS

Bioactive Agent is used herein to refer to a compound or entity thatalters, inhibits, activates, or otherwise affects biological or chemicalevents. For example, bioactive agents may include, but are not limitedto, osteogenic or chondrogenic proteins or peptides (including theosteoinductive or growth factors described above and synthetic andrecombinant version of these, including recombinant human bonemorphogenetic protein (rhBMP-2), available from Medtronic, Inc., asINFUSE® Bone Graft, and bone morphogenetic protein-7 (rhBMP-7 orOsteogenic Protein 1), available from Olympus Biotech Corp. in its OP-1products), anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors, hormones,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand antiadhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, angiogenic factors, anti-secretory factors, anticoagulantsand/or antithrombotic agents, local anesthetics, ophthalmics,prostaglandins, anti-depressants, anti-psychotic substances,anti-emetics, and imaging agents. In certain embodiments, the bioactiveagent is a drug. Bioactive agents further include RNAs, such as siRNA,and osteoclast stimulating factors. In some embodiments, the bioactiveagent may be a factor that stops, removes, or reduces the activity ofbone growth inhibitors. In some embodiments, the bioactive agent is agrowth factor, cytokine, extracellular matrix molecule or a fragment orderivative thereof, for example, a cell attachment sequence such as RGD.A more complete listing of bioactive agents and specific drugs suitablefor use in the present invention may be found in “PharmaceuticalSubstances: Syntheses, Patents, Applications” by Axel Kleemann andJurgen Engel, Thieme Medical Publishing, 1999; the “Merck Index: AnEncyclopedia of Chemicals, Drugs, and Biologicals”, Edited by SusanBudavari et al., CRC Press, 1996; and the United StatesPharmacopeia-25/National Formulary-20, published by the United StatesPharmcopeial Convention, Inc., Rockville Md., 2001, each of which isincorporated herein by reference.

Biocompatible, as used herein, is intended to describe materials that,upon administration in vivo, do not induce undesirable long-termeffects.

Bone as used herein refers to bone that is cortical, cancellous orcortico-cancellous of autogenous, allogenic, xenogenic, or transgenicorigin.

Bone particles herein refers to relatively small bone pieces such asfibers, bundles of loosely connected fibers, threads, narrow strips,thin sheets, chips, shards, particles, powders, other shapes, etc., thatpossess regular, irregular, or random geometries and which can beseparated form each other to any desired extent. While descriptionherein may be made specifically to bone fibers in some embodiments, asnoted it is to be appreciated that bone fibers are merely one type ofparticle that may be used with the implantable compositions disclosedherein.

Demineralized, as used herein, refers to any material generated byremoving mineral material from tissue, e.g., bone tissue. In certainembodiments, the demineralized compositions described herein includepreparations containing less than 8%, less than 5%, or less than 1%calcium by weight. Partially demineralized bone (e.g., preparations withgreater than 5% calcium by weight but containing less than 100% of theoriginal starting amount of calcium) is also considered within the scopeof the invention. In some embodiments, demineralized bone has less than95% of its original mineral content. Demineralized is intended toencompass such expressions as “substantially fully demineralized,”“substantially demineralized,” “partially demineralized,” and “fullydemineralized.”

Demineralized bone matrix or DBM, as used herein, refers to any materialgenerated by removing mineral material from bone tissue. In someembodiments, the DBM compositions as used herein include preparationscontaining less than 8%, less than 5%, or less than 1% calcium byweight. Partially demineralized bone (e.g., preparations with greaterthan 5% calcium by weight but containing less than 100% of the originalstarting amount of calcium) are also considered within the scope of theinvention.

Osteoconductive is used herein to refer to the ability of anon-osteoinductive substance to serve as a suitable template orsubstance along which bone may grow.

Osteogenic is used herein to refer to the ability of an agent, material,or implant to enhance or accelerate the growth of new bone tissue by oneor more mechanisms such as osteogenesis, osteoconduction, and/orosteoinduction.

Osteoinductive, as used herein, refers to the quality of being able torecruit cells from the host that have the potential to stimulate newbone formation. Any material that can induce the formation of ectopicbone in the soft tissue of an animal is considered osteoinductive. Mostosteoinductive materials induce bone formation in athymic rats whenassayed according to the method of Edwards et al., “Osteoinduction ofHuman Demineralized Bone: Characterization in a Rat Model,” ClinicalOrthopaedics & Rel. Res., 357:219-228, December 1998, incorporatedherein by reference. In other instances, osteoinduction is considered tooccur through cellular recruitment and induction of the recruited cellsto an osteogenic phenotype.

Osteoinductivity score, as used herein, refers to a score ranging from 0to 4 as determined according to the method of Edwards et al. (1998) oran equivalent calibrated test. In the method of Edwards et al., a scoreof “0” represents no new bone formation; “1” represents 1%-25% of theimplant being involved in new bone formation; “2” represents 26-50% ofthe implant being involved in new bone formation; “3” represents 51%-75%of the implant being involved in new bone formation; and “4” representsgreater than 75% of the implant being involved in new bone formation. Inmost instances, the score is assessed 28 days after implantation.However, the osteoinductivity score may be obtained at earlier timepoints, such as 7, 14, or 21 days following implantation. In theseinstances it may be desirable to include a normal DBM control such asDBM powder without a carrier, and, if possible, a positive control suchas BMP. Occasionally, osteoinductivity also may be scored at latertimepoints, such as 40, 60, or even 100 days following implantation.Percentage of osteoinductivity refers to an osteoinductivity score at agiven time point expressed as a percentage of activity of a specifiedreference score.

DETAILED DESCRIPTION I. Implantable Compositions

In various embodiments, the implantable compositions of the presentdisclosure may include a delivered material, and a carrier including agel base and a hydrating agent. The delivered material may comprise orcontain one or more bioactive compounds and/or biocompatible compounds.The water content of the carrier may be about 5% or less by weight ofthe implantable composition.

The inventors have determined that replacing the water in hydrogelcarriers with a substitute, nonaqueous hydrating agent can increase theshelf life of an implantable composition, and can alter the handlingcharacteristics of the implantable composition. It is believed that thepresence of water in hydrogel carriers may have a deleterious effect oncomponents often present the implantable material, particularly overtime. For example, in some implants, DBM, or other material comprisingor containing water-sensitive materials, may be provided in the hydrogelcarrier. Such water-sensitive materials may comprise, for example,proteins, growth factors, other biomaterials, bioglass, tricalciumphosphate, or a resorbable polymer. The water in the hydrogel maydegrade such water-sensitive materials over time, thereby adverselyaffecting the stability and/or shelf-life of the implant.

As an alternative to water, other liquid wetting or hydrating agentshaving multiple negatively charged moieties may be used to solubilize orotherwise disperse the polymeric base material of a hydrogel. In thismanner, the adverse effects of water on the stability of implantableproducts formed using hydrogels may be eliminated, reduced, ormitigated.

A. Bioactive and Biocompatible Compounds

The delivered material may be any material comprising or suitable forcarrying bioactive compounds and/or biocompatible compounds. Dependingon the delivered material selected, such bioactive compounds and/orbiocompatible compounds may be endogenous to the material or may beexogenously added to the material. Any suitable material may be used,including, but not limited to, bone, other tissue, hydroxyapatite,ceramics, calcium sulfate, calcium phosphates, tricalcium phosphate,bioactive glasses, bioactive agents, other materials, and combinationsof these.

a. Bioactive Compounds

In some embodiments, the implantable compositions of the presentdisclosure may include a delivered material comprising or containingbioactive agents, which may include bioactive compounds and/orbiocompatible compounds.

Bioactive compounds may be endogenously present in the material carriedby the hydrogel or exogenously added to the material carried by thehydrogel. Suitable bioactive compounds for the inventive compositionsmay include, for example, bone, other tissue, growth factors,glycoproteins, and/or other bioactive materials suitable for use in therepair of tissue or bone, such as chondroitin sulfate, and may includebioactive agents. Suitable growth factors may include recombinant humanbone morphogenetic protein (rhBMP-2), available from Medtronic, Inc., asINFUSE® Bone Graft; bone morphogenetic protein-7 (rhBMP-7 or OsteogenicProtein 1), available from Olympus Biotech Corp. in its OP-1 products;other growth factors; anti-AIDS substances; anti-cancer substances;antibiotics; immunosuppressants; anti-viral substances; enzymeinhibitors; hormones; neurotoxins; opioids; hypnotics; anti-histamines;other materials; and combinations of these. Suitable growth factors alsomay include members of the TGF-β, IGF, and BMP protein families. Asnoted, the bioactive compounds may be endogenously present in a materialof the implantable compositions, as in the case of bone or bone-derivedelements (e.g., demineralized bone), may be exogenously added to theimplantable composition, may be combinations of endogenous and exogenousbioactive compounds, etc.

Biocompatible Compounds

In various embodiments, in addition to or in lieu of bioactive agents,the delivered material may comprise or contain one or more biocompatiblecompounds. Suitable biocompatible compounds may include, for example,bioactive glass or bioglass, hydroxyapatite, ceramics, calcium sulfates,calcium phosphates, tricalcium phosphate, polymers (e.g., porouspolymers, resorbable polymers), 1,2,3-trichloropropane, metal particles(e.g., titanium, shape-memory alloys), and/or other biocompatiblematerials suitable for use in the repair of tissue or bone.

c. Bone

In a specific embodiment, the delivered material may be bone or bonederived elements. The bone may be a bioactive agent, or not. The bonemay be obtained utilizing methods well known in the art, e.g., allogenicdonor bone. Generally, the bone can be nondemineralized, partiallydemineralized (including surface demineralized), demineralized,deorganified, anorganic, or combinations thereof.

The bone or bone-derived elements can be readily obtained from donorbone by various suitable methods, e.g., as described in U.S. Pat. No.6,616,698, incorporated herein by reference. Autogenic, allogenic,xenogenic, or transgenic bone can be used. Suitable types of xenogenicbone may include porcine, equine, or bovine. The bone can be cortical,cancellous or corticocancellous. In specific embodiments describedherein, the bone is cortical allogenic bone, e.g., femur, tibia, fibula,radius, ulna, etc.

The bone suitable for use in the implantable compositions herein are notlimited to any size or configuration of bone to be treated. Boneutilized as the starting, or stock, material may range in size fromrelatively small pieces of bone to bone of such dimensions as to berecognizable as to its anatomical origin to whole bone. For example, thestock material may range from fragments to whole bone and the formedbone composition may range from particles to fibers to plates. In someembodiments, the whole bone may be partially subdivided, such as intostrips, for further processing by methods disclosed herein.

Separating the bone (or separation), also referred to as particulation,refers to subdivision of the bone. Separating the bone may provide spaceand surface area for cellular activity (osteoconductivity). Separatingthe bone may further expose bone inducing/facilitating proteins toinduce bone regeneration (osteoinductivity). Generally, separation maybe done in a manner such that the bone subdivides in a manner generallyconsistent with natural morphology. In some embodiments, separation maybe used to subdivide the bone into bone particles, fibers, plates, orother desired configuration and size. In some embodiments, separationmay be used to subdivide the bone into bone fibers. Thickness and widthof the fibers may be generally equal and length of the fibers may be upto the length of the stock material.

Separation may be done by any suitable technique. For example, machinesor instruments that cut, shred, mill, extrude, and/or chop may be used.Examples of suitable equipment may use a sharp bit or blade on or intothe bone to cut or mill the bone, including by cutting away materialthat is not in the desired configuration (such as material that is notfibrous). For example, mills, including ball mills, impact mills,grating mills, shearing mills, and cutting mills, may be used.

d. Demineralized Bone

In some embodiments, the delivered material may comprise demineralizedbone. DBM preparations have been used for many years in orthopedicmedicine to promote the formation of bone. For example, DBM has founduse in the repair of fractures, in the fusion of vertebrae, in jointreplacement surgery, and in treating bone destruction due to underlyingdisease such as a bone tumor. DBM is has been shown to promote boneformation in vivo by osteoconductive and osteoinductive processes. Theosteoinductive effect of implanted DBM compositions results from thepresence of active growth factors present on the isolated collagen-basedmatrix. These factors include members of the TGF-β, IGF, and BMP proteinfamilies. Particular examples of osteoinductive factors include TGF-β,IGF-1, IGF-2, BMP-2, BMP-7, parathyroid hormone (PTH), and angiogenicfactors. Other osteoinductive factors such as osteocalcin andosteopontin are also likely to be present in DBM preparations as well.There are also likely to be other unnamed or undiscovered osteoinductivefactors present in DBM.

For use in the compositions of the present invention, the bone may bedemineralized in any suitable manner. Generally, demineralizationprocedures remove the inorganic mineral component of bone by employingacid solutions, referred to as an acid treatment. Such procedures arewell known in the art, see for example, Reddi et al., Proceeding of theNational Academy of Sciences of the United States of America 69, pp.1601-1605 (1972), incorporated herein by reference. The strength of theacid solution, the shape and size of the bone and the duration of thedemineralization procedure influence the extent of demineralization andmay be selected for the particular application. Generally speakinglarger bone portions as compared to small particles require more lengthyand vigorous demineralization. For example, small fibers may bedemineralized within approximately 3 hours while large fibers may bedemineralized within approximately 4 hours. Guidance for specificparameters for demineralization and for the demineralization ofdifferent size bone can be found in U.S. Pat. No. 5,846,484, Harakas,Clinical Orthopaedics and Related Research, pp 239-251(1983) andLewandrowski et al., Journal of Biomedical Materials Research, 31, pp.365-372 (1996), and U.S. Pat. Nos. 6,189,537 and 6,305,379, each ofwhich is incorporated by reference herein. U.S. patent application Ser.No. 12/849,054, filed Aug. 3, 2010, discloses suitable methods fordemineralization that may be used in accordance with the presentinvention and is herein incorporated by reference in its entirety.

After acid treatment, the demineralized bone may be rinsed with sterilewater for injection or sterile purified water to remove residual amountsof acid.

B. Carriers

In illustrative embodiments, the delivered material of the implantablecompositions may be provided in a carrier. Generally, a carrier may beemployed to facilitate in vivo use and/or formation of a particularconfiguration of the implantable configuration.

In some embodiments, the carrier may include a non-aqueous, orsubstantially non-aqueous, gel. As used herein, the term gel may referto a solid, polymeric material (hereinafter, “gel base”) that is atleast partially solubilized in, or otherwise dispersed in, a liquid(hereinafter, “hydrating agent”). Suitable gel bases may includechitosan, alginate, hyaluronic acid, polyvinyl alcohols, polyvinlypyrolidone, dextran, carboxymethyl cellulose, collagen, starches,gelatin, extracted proteins, starches, any other water soluble polymericmaterials, and/or combinations thereof. Specific forms of hyaluronicacid as carriers for demineralized bone are described in U.S. Pat. No.6,030,635, incorporated by reference herein. Suitable hydrating agentsmay include any liquids which can be substituted for water to at leastpartially solubilize a gel base. For example, hydrating agents mayinclude liquid polyhydroxy compounds such as glycerol, low molecularweight polyethylene glycol, high molecular polyethylene glycol, and/orany other liquids having multiple negatively charged moieties, andcombinations thereof. Suitable polyhydroxy compounds are listed in U.S.Pat. No. 5,290,558, previously incorporated by reference herein.

C. Additives

Optionally, other additives, including bioactive agents, may be includedin the implantable compositions disclosed herein. It will be appreciatedthat the amount of additive used may vary depending upon the type ofadditive, the specific activity of the particular additive preparationemployed, and the intended use of the composition. The desired amountmay be readily determinable by the user. Any of a variety of medicallyand/or surgically useful optional substances can be incorporated in, orassociated with, the osteoinductive factors either before, during, orafter preparation of the osteogenic composition.

In certain embodiments, the additive is adsorbed to or otherwiseassociated with the implantable composition. The additive may beassociated with the implantable composition through specific ornon-specific interactions, or covalent or noncovalent interactions.Examples of specific interactions include those between a ligand and areceptor, an epitope and an antibody, etc. Examples of nonspecificinteractions include hydrophobic interactions, electrostaticinteractions, magnetic interactions, dipole interactions, van der Waalsinteractions, hydrogen bonding, etc. In certain embodiments, theadditive is attached to the implantable composition, for example, to thecarrier, using a linker so that the additive is free to associate withits receptor or site of action in vivo. In other embodiments theadditive is either covalently or non-covalently attached to the carrier.In certain embodiments, the additive may be attached to a chemicalcompound such as a peptide that is recognized by the carrier. In anotherembodiment, the additive is attached to an antibody, or fragmentthereof, that recognizes an epitope found within the carrier. In certainembodiments at least additives are attached to the implantablecomposition. In other embodiments at least three additives are attachedto the implantable composition. An additive may be provided within theimplantable composition in a sustained release format. Thus, delivery ofa growth factor in an implantable composition may include micro- ornano-encapsulation. For example, the additive may be encapsulated withinbiodegradable nanospheres, microspheres, etc. In this method, a growthfactor (or other bioactive agent) is first encapsulated in a suitablematerial, such as a bioresorbable polymer or hydrogel micro- ornano-particle. The encapsulated growth factor then is immobilized ontoor into the implantable composition. The bioresorbable polymer/hydrogelmay be designed to control release kinetics of the growth factor.

It will be understood by those skilled in the art that the lists ofoptional substances herewith included are not intended to be exhaustiveand that other materials may be admixed with the delivered materialswithin the practice of the present invention.

Radiopaque substances may be added to impart radiopacity to thecomposition. Examples of substances imparting radiopacity include forexample, bone particles that have not been fully demineralized,including mineralized cancellous bone, mineralized cortical bone, andpartially demineralized bone in solid, particle, or fiber form; Barium-and Iodine-containing compounds or compositions, e.g., Barium Sulfateand Barium Sulfate for Suspension; Iopanoic Acid; and the like. U.S.Pat. No. 5,676,146 discusses radiopaque implantable materials and isherein incorporated by reference in its entirety.

Development of a vasculature around the implant site may also bebeneficial in forming new bone and/or cartilage tissue. Angiogenesis maybe a contributing factor for the replacement of new bone and cartilagetissues. In certain embodiments, angiogenesis may be promoted so thatblood vessels are formed at the site to allow efficient transport ofoxygen and other nutrients and growth factors to the developing bone orcartilage tissue. Thus, angiogenesis promoting factors may be includedin the osteoimplant to increase angiogenesis in that region. Forexample, class 3 semaphorins, e.g., SEMA3, controls vascularmorphogenesis by inhibiting integrin function in the vascular system,Serini et al., Nature, (July 2003) 424:391-397, incorporated herein byreference, and may be included in the osteoimplant. Vascular EndothelialGrowth Factor (VEGF) and other cystine-knot growth factors may be usedas well.

III. Methods of Preparing

Methods for preparing the implantable compositions of the presentinvention may include the substitution of a nonaqueous, liquid hydratingagent for water in a carrier, such as in a hydrogel material. Thistechnique is useful for preparing non-aqueous solutions of materials insolvents that the materials do not directly dissolve in. For example,carboxy methylcellulose will not dissolve in glycerol even if theglycerol is heated. But carboxy methylcellulose, if first dissolved inwater, can be converted to a glycerol solution by adding glycerol to thewater solution of carboxy methylcellulose, then removing the water by adrying process, preferably involving vacuum. The substitution may beachieved by dissolving a gel base in water to form a mixture, adding ahydrating agent to the mixture, and reducing the water content of themixture. Not to be limited in theory, it is believed that in thepresence of water, the gel base has an “open” structure, whichfacilitates replacement of the O—H bonds of water with the hydroxidegroups of a liquid hydrating agent as the water is removed duringdrying.

Another advantage of the process is that it allows the incorporation ofa heat sensitive substance, such as BMP-2, into the hydrogel. Many basegel materials can only dissolve in water (or other solvent) by theapplication of heat. Starch, for example, has to be boiled in water todissolve. With the solvent substitution technique, BMP-2 can bedissolved in water (at room temperature), then added to glycerol, andthen the water/BMP-2/glycerol mixture can be combined with the hydratedstarch. Lyophilization (or mild vacuum drying) will serve to incorporatethe BMP-2 and glycerol with the starch while eliminating the water;resulting in a stable starch/glycerol/BMP-2 gel that could be used forbone grafting applications.

FIG. 1 illustrates a method of preparing an implantable composition inaccordance with one embodiment of the present disclosure. In oneembodiment, the method 10 may include combining a gel base with water toform a mixture [block 12]. Following the step of block 12, a deliveredmaterial may be combined with the gel base-water mixture of block 12[block 14]. The delivered material may comprise or include one or morebioactive and/or biocompatible compounds. The method may then includeadding a hydrating agent to the mixture of block 14 [block 16].Alternatively, or additionally, the hydrating agent may be added in step12 (e.g., in the form of a water-hydrating agent mixture). The hydratingagent may be added such that the hydrating agent is present in an amountranging from about 1% to about 90% by weight of total liquid in themixture (i.e., the water and the hydrating agent). Alternatively, thehydrating agent may be present in an amount ranging from about 1% toabout 50% by weight of the total liquid in the mixture. In a furtheralternative, the hydrating agent may be present in an amount rangingfrom about 1% to about 10% by weight of the total liquid in the mixture.

Following the step of block 14, the water content of the mixture ofblock 16 may be reduced, thereby leaving a composition [block 18].Reduction of the water content may be achieved by drying techniques. Inone embodiment, reduction of the water content is accomplished bylyophilization or by vacuum drying. The water content may be reduced toabout 5% or less by weight of the composition. Alternatively, the watercontent may be reduced to about 3% or less by weight of the composition.In a further alternative, the water content may be reduced to about 1%or less by weight of the composition.

In an alternative to the embodiment of FIG. 1, the addition of thedelivered material (the step of block 14) may occur concomitantly withand/or subsequent to the step of block 16 and/or the step of block 18.

A. Formulation

The relative amounts of gel base, hydrating agent, and deliveredmaterial in the implantable compositions prepared utilizing the methodsof the present disclosure may be selected such that the compositionexhibits physical properties (e.g., deformability, viscosity, etc.) thatare desired for the particular clinical application. For example, byvarying the relative amounts of the components, the implantablecompositions may be formulated to be settable, moldable, injectable, orthe like.

In various embodiments, the implantable compositions may be formulatedsuch that compositions include about 20 to about 90 percent by weightgel base, about 10 to about 80 percent by weight hydrating agent, andabout 10 to about 50 weight percent delivered material. Alternatively,the relative weight percentages of the components of the implantablecomposition may have any range suitable for the particular clinicalapplication.

In illustrative embodiments, the implantable compositions preparedutilizing the methods of the present disclosure may exhibit excellentsurgical handling properties, which can range from a viscous liquid forinjection (high hydrating agent content), to a hard formable materialfor defined hard tissue defects (low hydrating agent content), while notcausing the premature degradation of contained bioactive materialsassociated with hydrogel carriers. Moreover, the compositions mayexhibit great resistance to surgical irrigation and absence of tendencyfor crystallization, without a loss of osteoinductivity relative tocompositions having a hydrogel carrier.

IV. Uses

The implantable compositions of the present disclosure may be used for avariety of orthopedic procedures where they participate in a bonehealing/repair process through one or more mechanisms such asosteogenesis, osteoinduction, and osteoconduction (e.g., tissue graftingof either bone or soft tissue defects). The implantable compositions maybe used as is, or formed into a variety of product types, such as gels,putties, pastes, cakes, sheets, etc. The implantable compositions may beused as an injectable material to fill graft sites, packed into defects,or packed into spinal cages or other devices that can hold a graftmaterial, and may swell upon hydration to expand into a site. Theimplantable compositions also may be used for drug delivery or othercontrolled release applications. The implantable compositions canoptionally be admixed with one or more substances such as adhesives,fillers, plasticizers, flexibilizing agents, biostatic/biocidal agents,surface active agents, binding and bonding agents, and the like, priorto, during, or after shaping the particles into a desired configuration.Suitable adhesives, binding agents and bonding agents include acrylicresins, cellulosics, bioresorbable polymers such as polyesters,polycarbonates, polyarylates and polyfomarates. Specifically, tyrsine,polycarbonates, tyrosine polyarylates, polyglycolides, polylactides,glycolide-lactide copolymer, etc. Suitable fillers include bone powder,demineralized bone powder, hydroxyapatite, etc. Suitable plasticizersand flexibilizing agents include liquid polyhydroxy compounds such asglycerol, monacetin, diacetin, etc. Suitable biostatic/biocidal agentsinclude antibiotics, providone, sugars, etc. Suitable surface-activeagents include the biocompatible nonionic, cationic, anionic andamphoteric surfactants. The implantable compositions may be combinedwith a polymer to form a biocomposite. Suitable materials for preparingbiocomposites are disclosed in U.S. Patent Publication Nos.2007/0191963, 2006/0216323, and 2005/0251267, U.S. Pat. Nos. 6,696,073,6,478,825, 6,440,444, and 6,294,187, all herein incorporated byreference in their entireties for all purposes. The implantablematerials of the present invention may be delivered alone, or insideother devices, such as cages, frames, or delivery vehicles, includingbut not limited to polymeric, collagen, and/or other organic orsynthetic resorbable or nonresorbable fabrics or coverings. U.S. Pat.Nos. 7,323,193, 7,163,691, 6,863,694, 6,808,585. 6,616,698, 6,599,520,6,436,138, 5,676,146, 5,510,396, 5,507,813, 5,484,601, 5,439,684,5,405,390, 5,314,476, 5,298,254, 5,290,558, 5,284,655, 5,236,456,5,073,373, U.S. Patent Application Publications Nos. 2007/0098756,2007/0110820; 2007/0154563; 2009/0130173, and 2009/0192474 and U.S.patent application Ser. Nos. 12/171,168; 12/205,539; 12/140,062;12/267,985; 12/140,025; 12/267,985; 12/254,619; 61/108,350, 61/152,057;61/154,673; 61/154,679, and 61/154,689 also are herein incorporated byreference in their entireties.

The implantable compositions of the present disclosure may be subjectedto a configuring step to form an implant. The configuring step can beemployed using conventional equipment known to those skilled in the artto produce a wide variety of geometries, e.g., concave or convexsurfaces, stepped surfaces, cylindrical dowels, wedges, blocks, screws,and the like. A surgically implantable material fabricated fromelongated bone particles that have been demineralized, which may beshaped as a sheet, and processes for fabricating shaped materials fromdemineralized bone particles are disclosed in U.S. Pat. Nos. 5,507,813and 6,436,138, respectively, the contents of which are incorporated byreference herein. Suitable sheets include those sold under the tradename Grafton® DBM Flex, which must be wetted/hydrated prior to use to beuseful for implantation. Such sheets have recently been reported aseffective in seeding human bone marrow stromal cells, which may beuseful in the repair of large bone defects. Kasten et al., “Comparisonof Human Bone Marrow Stromal Cells Seeded on Calcium-DeficientHydroxyapatite, Betatricalcium Phosphate and Demineralized Bone Matrix,”Biomaterials, 24(15):2593-603, 2003.

The implantable compositions of the present disclosure may assume adetermined or regular form or configuration such as a sheet, plate,disk, tunnel, cone, or tube, etc. Prefabricated geometry may include,but is not be limited to, a crescent apron for single site use, anI-shape to be placed between teeth for intra-bony defects, a rectangularbib for defects involving both the buccal and lingual alveolar ridges,neutralization plates, reconstructive plates, buttress plates,T-buttress plates, spoon plates, clover leaf plates, condylar plates,compression plates, bridge plates, or wave plates. Partial tubular aswell as flat plates can be fabricated from the osteoimplant. Such platesmay include such conformations as, e.g., concave contoured, bowl shaped,or defect shaped. The implantable compositions can be machined or shapedby any suitable mechanical shaping means. Computerized modeling canprovide for the intricately-shaped three-dimensional architecture of animplantable compositions custom-fitted to the bone repair site withgreat precision.

V. Examples Example 1

Viscous chitosan solutions were prepared as follows:

100 ml of deionized water was measured out. 3 g of citric acid anhydrouswas added to the deionized water. 3 g of chitosan was added and themixture stirred by hand until dissolved and a viscous solution resulted.Following the addition of chitosan, a hydrating agent (glycerol, highPEG (35,000 MW), low PEG (20,000 MW), or PEG liquid (600 MW)), and abioactive component (re-mineralized DBM powder or demineralized powder)were added to the viscous solution. Table 1 below describes amounts ofhydrating agent and bioactive component added to each preparation.Following the addition of hydrating agent and bioactive component, thecompositions were frozen at −70° C. for about 6 hours, then lyophilizedusing a program that started at −54° C. and warmed to 35° C. The totalcycle length was 48 hours, but the material can be dried usingalternative temperatures and cycle lengths, for example using a 35degree heat cycle for 24 hours.

TABLE 1 Amount/Prep. # Component A B C D E F G H I J K L Viscouschitosan solution   16 g   16 g   16 g   14 g   14 g   14 g   14 g   14g   14 g  16 g  16 g  16 g Glycerol 3.32 g 5.16 g 7.00 g 7.04 g 6.03 g5.01 g 4.18 g 1.45 g — 3.61 g  5.35 g  7.11 g  PEG - 35,000 MW — — — — —— — — — 0.5 g 0.5 g 0.5 g PEG - 20,000 MW — — — — — — — — — 0.5 g 0.5 g0.5 g PEG liquid - 600 MW — — — — 1.04 g 2.03 g 3.63 g 6.11 g 7.13 g — —— Re-mineralized DBM   3 g — — — — — — — —   3 g   3 g   3 g powderDemineralized powder — — — 0.71 g 0.71 g 0.71 g 0.71 g 0.71 g 0.71 g — ——

Table 2 includes the observations made with respect to each of thepreparations of Table 1.

Preparation number Properties A Flexible adhesive cake uponlyophilization; swells in water by about 3x; does not disintegrate BFlexible adhesive cake upon lyophilization; swells in water by about 3x;does not disintegrate C Flexible adhesive cake upon lyophilization;swells in water by about 3x; does not disintegrate; less slimy thanlower glycerol content preparations D Flexible adhesive cake uponlyophilization; swells in water by about 3x; does not disintegrate EFlexible adhesive cake upon lyophilization; swells in water by about 3x;does not disintegrate F Flexible adhesive cake upon lyophilization;swells in water by about 3x; does not disintegrate G Flexible adhesivecake upon lyophilization; swells in water by about 3x; does notdisintegrate H Flexible adhesive cake upon lyophilization; swells inwater by about 3x; very weak upon handling I Flexible adhesive cake uponlyophilization; disintegrates in water J Flexible adhesive cake uponlyophilization; swells in water by about 3x; does not disintegrate KFlexible adhesive cake upon lyophilization; swells in water by about 3x;does not disintegrate L Flexible adhesive cake upon lyophilization;swells in water by about 3x; does not disintegrate

It was observed that the compositions with viscous liquid properties usea higher polyhydroxy content and may be used as injectable materials tofill graft sites. It was also observed that compositions having gel orflexible properties may be packed into defects, packed into deliveryvehicles such as spinal cages or other implants that can hold a graftmaterial, and will generally swell upon hydration to expand in a site.It was further observed that compositions exhibiting hard materialproperties (higher loading of bioactive material such as DBM) may bemolded into specific shapes that may facilitate use in spinal cages asfillers, deep screw holes, etc. and will swell upon hydration to fill asite. Such compositions further may be useful in controlled release drugdelivery applications.

Example 2

Samples were prepared as follows. A starch was hydrated with water. Thehydrated starch was mixed with glycerol. Thereafter, DBM particles (sameas Gel; 106 to 500 microns) were added to the hydrated starch-glycerolmixture. The resulting product was lyophilized. Lyophilization allowedthe glycerol to nearly completely replace the water in the starch.Testing of a sample containing 56% glycerol by weight showed a watercontent of only 0.53%, making the formulation about 29% bone and 71%nonaqueous carrier.

Handling Properties

The handling properties of the resulting compositions at least partiallydepend on the amount of glycerol in the composition. For example, asample composition containing 55% glycerol was flowable, while a samplecontaining 49% glycerol had stiff, putty-like qualities.

Irrigation

The samples were tested for resistance to irrigation compared toglycerol gel and paste analogues [We should include details relating tothe specifics of those compositions. In general, Gel and Putty are 20 to30% bone and 70 to 80% glycerol. Gel is flowable, Putty is stiffer.However, Putty has long fibers that tangle together to make a cohesiveproduct, and Gel is just particles. In comparison, the starch-glycerolproducts showed great resistance to irrigation, making it suitable forother applications.

Stability

A sample composition containing 49% glycerol was stored in ambientconditions for 5 months. After 5 months, its handling properties weresubstantially unchanged and there was no sign of crystallization.Additionally, there was no sign of mold grown, which would be expectedwith a water-based formulation.

Example 3

Osteoinductivity testing was carried out on 4 different samples, all ofwhich were made from the same R&D donor DBM. The samples were: controlDBM powder; DBM in starch-glycerol carrier containing about 49% glycerolto yield a stiff paste; alginate-glycerol DBM formulation; and ahyaluronic acid-starch DBM formulation. The latter two materials weremade up using the same procedures as the starch-glycerol formulation. Asmall preliminary athymic model study was designed and carried out toobserve any preliminary effects of these 3 anhydrous carriers onosteoinductivity. According to histology and pCT Bone/Nodulecalculations, all three carriers performed well (with typical animalvariability) in comparison to the control, which itself scored anaverage of 2.5. The same lot of donor DBM had been used previouslyyielding an osteoinductivity score of 2, so was considered to be amid-range control. Starch-glycerol and alginate-glycerol performedidentically with an average osteoinductivity score of 1.75, whilehyaluronic acid-glycerol performed better with an averageosteoinductivity score of 2.00 Table 3 below summarizes therandomization and calculations.

TABLE 3* Study # Animal # LIM % Bone OI Score RIM % Bone OI Score ImplTime 10-004 1 CON 38.0 3 STG 32.3 3 28 10-004 2 ALG 32.2 3 HAG 27.0 3 2810-004 3 STG 6.5 1 ALG 3.1 1 28 10-004 4 HAG 17.0 2 CON 29.0 3 28 10-0045 ALG 16.2 2 STG 37.3 2 H 28 10-004 6 CON 25.3 2 ALG 6.3 1 H 28 10-004 7STG 14.7 1 H HAG 13.1 2 28 10-004 8 HAG 5.6 1 CON 17.1 2 28 *STG(Starch/Glycerol), ALG (Alginate/Glycerol), HAG (HyaluronicAcid/Glycerol) Athymic study results in comparison to the control bone(no carrier present) (H denotes it was scored in the higher realm of theclassification)

The handling characteristics are vastly improved and in the currentlytested paste formulation, the shelf life lengthened considerably withthe elimination of water and the presence of glycerol to act as anantimicrobial.

The invention claimed is:
 1. A method for preparing an implantablecomposition comprising: (i) providing a gel base comprising dextran;(ii) adding water, anhydrous citric acid and a hydrating agent to thegel base to form a mixture; (iii) reducing the water content of themixture; and (iv) adding a delivered material before, during, and/orafter step (ii) or (iii); wherein the water content is reduced to about5% or less by weight of the implantable composition.
 2. The method ofclaim 1, wherein the delivered material includes or comprises abioactive compound and/or a biocompatible compound.
 3. The method ofclaim 1, wherein the gel base further comprises chitosan, alginate, orcombinations thereof.
 4. The method of claim 1, wherein the hydratingagent comprises a polyhydroxy compound.
 5. The method of claim 4,wherein the polyhydroxy compound is selected from the group consistingof glycerol, low molecular weight polyethylene glycol, high molecularweight polyethylene glycol, or combinations thereof.
 6. The method ofclaim 1, wherein the step of adding a delivered material comprisesadding a bioactive material.
 7. The method of claim 6, wherein thebioactive material comprises at least one osteogenic and/orosteoinductive material.
 8. The method of claim 7, wherein theosteogenic and/or osteoinductive material comprises demineralized bone.9. The method of claim 1, wherein the step of reducing the water contentcomprises subjecting the composition to lyophilization.
 10. The methodof claim 9, wherein the water content is reduced to about 3% or less byweight of the implantable composition.
 11. The method of claim 10,wherein the water content is reduced to about 1% or less by weight ofthe implantable composition.
 12. An implantable composition comprising:a delivered material, and a carrier comprising: a gel base comprisingdextran; anhydrous citric acid; and a hydrating agent; wherein a watercontent of the implantable composition is less than about 5% by weightof the composition.
 13. The implantable composition of claim 12, whereinthe delivered material includes or comprises a bioactive compound and/ora biocompatible compound.
 14. The implantable composition of claim 13,wherein the bioactive material comprises at least one osteogenic and/orosteoinductive material.
 15. The implantable composition of claim 14,wherein the osteogenic and/or osteoinductive material comprisesdemineralized bone.
 16. The implantable composition of claim 12, whereinthe gel base further comprises chitosan, alginate, hyaluronic acid,polyvinyl alcohol, polyvinyl pyrolidone, carboxymethyl cellulose,collagen, gelatin, extracted proteins, a starch, or combinationsthereof.
 17. The implantable composition of claim 16, wherein the gelbase further comprises chitosan, alginate, or combinations thereof. 18.The implantable composition of claim 12, wherein a heat sensitivesubstance is incorporated into the hydrogel.
 19. The implantablecomposition of claim 18, where the heat sensitive substance is BMP-2.